title: Targeting of the Hedgehog signal transduction pathway suppresses survival of malignant pleural mesothelioma cells in vitro.
abstract: OBJECTIVE
  The present study sought to determine whether the Hedgehog (Hh) pathway is active and regulates the cell growth of cultured malignant pleural mesothelioma (MPM) cells and to evaluate the efficacy of pathway blockade using smoothened (SMO) antagonists (SMO inhibitor GDC-0449 or the antifungal drug itraconazole [ITRA]) or Gli inhibitors (GANT61 or the antileukemia drug arsenic trioxide [ATO]) in suppressing MPM viability.
  
  
  METHODS
  Selective knockdown of SMO to inhibit Hh signaling was achieved by small interfering RNA in 3 representative MPM cells. The growth inhibitory effect of GDC-0449, ITRA, GANT61, and ATO was evaluated in 8 MPM lines, with cell viability quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell death was determined by annexinV/propidium iodide staining and flow cytometry.
  
  
  RESULTS
  SMO small interfering RNA mediated a two- to more than fivefold reduction of SMO and Gli1 gene expression as determined by real-time quantitative reverse-transcriptase polymerase chain reaction, indicating significant Hh pathway blockade. This was associated with significantly reduced cell viability (34% ± 7% to 61% ± 14% of nontarget small interfering RNA controls; P = .0024 to P = .043). Treating MPM cells with Hh inhibitors resulted in a 1.5- to 4-fold reduction of Gli1 expression. These 4 Hh antagonists strongly suppressed MPM cell viability. More importantly, ITRA, ATO, GANT61 induced significant apoptosis in the representative MPM cells.
  
  
  CONCLUSIONS
  Hh signaling is active in MPM and regulates cell viability. ATO and ITRA were as effective as the prototypic SMO inhibitor GDC-0449 and the Gli inhibitor GANT61 in suppressing Hh signaling in MPM cells. Pharmaceutical agents Food and Drug Administration-approved for other indications but recently found to have anti-Hh activity, such as ATO or ITRA, could be repurposed to treat MPM.
authors.affiliation: Thoracic Surgery Section, Division of Cardiothoracic Surgery, Department of Surgery, Sylvester Comprehensive Cancer Center, Leonard M. Miller School of Medicine, University of Miami, Miami, Fla.
authors.forename: Min
authors.initials: M
authors.lastname: You
authors.affiliation: Thoracic Surgery Section, Division of Cardiothoracic Surgery, Department of Surgery, Sylvester Comprehensive Cancer Center, Leonard M. Miller School of Medicine, University of Miami, Miami, Fla.
authors.forename: Javier
authors.initials: J
authors.lastname: Varona-Santos
authors.affiliation: Molecular Oncology Program, Department of Surgery, Sylvester Comprehensive Cancer Center, Leonard M. Miller School of Medicine, University of Miami, Miami, Fla.
authors.forename: Samer
authors.initials: S
authors.lastname: Singh
authors.affiliation: Molecular Oncology Program, Department of Surgery, Sylvester Comprehensive Cancer Center, Leonard M. Miller School of Medicine, University of Miami, Miami, Fla.
authors.forename: David J
authors.initials: DJ
authors.lastname: Robbins
authors.affiliation: Division of Oncology, Department of Medicine, Miami Veterans Affairs Medical Center, Sylvester Comprehensive Cancer Center, Leonard M. Miller School of Medicine, University of Miami, Miami, Fla.
authors.forename: Niramol
authors.initials: N
authors.lastname: Savaraj
authors.affiliation: Thoracic Surgery Section, Division of Cardiothoracic Surgery, Department of Surgery, Sylvester Comprehensive Cancer Center, Leonard M. Miller School of Medicine, University of Miami, Miami, Fla. Electronic address: dnguyen4@med.miami.edu.
authors.forename: Dao M
authors.initials: DM
authors.lastname: Nguyen
mesh_terms: D000813:Anilides / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D000077237:Arsenic Trioxide; D001152:Arsenicals / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D015972:Gene Expression Regulation, Neoplastic; D053823:Hedgehog Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017964:Itraconazole / Q000494:pharmacology; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008654:Mesothelioma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D000086002:Mesothelioma, Malignant; D058990:Molecular Targeted Therapy; D010087:Oxides / Q000494:pharmacology; D010997:Pleural Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D034622:RNA Interference; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D000072086:Smoothened Receptor; D013997:Time Factors; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D000071676:Zinc Finger Protein GLI1
chemical_list: D000813:Anilides; D000970:Antineoplastic Agents; D001152:Arsenicals; C551027:GANT 61; C400551:GLI1 protein, human; D053823:Hedgehog Proteins; C538724:HhAntag691; D010087:Oxides; D011725:Pyridines; D011743:Pyrimidines; D043562:Receptors, G-Protein-Coupled; C491023:SMO protein, human; D000072086:Smoothened Receptor; D014157:Transcription Factors; D000071676:Zinc Finger Protein GLI1; D017964:Itraconazole; D000077237:Arsenic Trioxide
keywords: 14; 29; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ATO; EGFR; FDA; Food and Drug Administration; GDC; GDC-0449; HPI; Hedgehog pathway inhibitor; Hh; IC(50); ITRA; MPM; MTT; PCR; PI; PTCH; RT; SMO; arsenic trioxide; drug concentration that mediates 50% inhibition of cell number or viability; epidermal growth factor receptor; hedgehog; itraconazole; mRNA; malignant pleural mesothelioma; messenger RNA; nontarget scramble control siRNA; patched; polymerase chain reaction; propidium iodide; reverse transcriptase/reverse transcribed; siNT; siRNA; siRNA targeting SMO; siSMO; small interfering RNA; smoothened